Ribosome biogenesis is an essential and complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three separate structural RNAs that are transcribed as a single precursor molecule. This precursor must be correctly modified, folded, processed and assembled with proteins to yield the two mature ribosomal subunits that comprise the functional ribosome. Severe mutations in this pathway are lethal. Minor perturbations are characterized by diseases including dyskeratosis congenital and some autoimmune diseases. The focus of the research in my lab has been to identify and characterize cis-acting elements and trans-acting factors critical for the processing events of pre-rRNA processing, and thus essential for cell growth and survival. Over the past year my lab has made progress on three fronts. First, we are using the genetics available in yeast, S.cerevisiae, to differentiate between two predicted structural models for an intramolecular interaction in pre-rRNA necessary for subsequent processing steps. Identification of the structural conformation of the precursor is essential for understanding how the structure affects ribosome biogenesis. Second, we are using biochemical methods to identify proteins that comprise the Xenopus U8 small nucleolar ribonucleoprotein particle (U8 snoRNP), an essential trans-acting factor required for accumulation of newly formed large ribosomal subunits. Third, we are examining the kinetics of pre-rRNA processing to learn more, at a mechanistic level, about the roles that snoRNPs, particularly U8, play in pre-rRNA processing. One focus in the lab involves a more detailed examination of our previously described a model for the mechanism by which U8 snoRNA may facilitate pre-rRNA processing in the Xenopus oocyte (1). This model predicted that formation of a specific intramolecular interaction in pre-rRNA should be critical for pre-rRNA processing. Because of the many different aspects of RNA processing addressed by this model and complexity of the Xenopus oocyte system, the yeast system was used to directly test this one aspect of the model. The feasibility of genetic and biochemical manipulations in yeast made it possible to assay the effects of point mutations in this region upon the ability to process pre-rRNA. Our early experiments in yeast unequivocally demonstrated that formation of this intramolecular interaction is critical for pre-rRNA processing (2, 3). Over the past year we have identified essential alterations in structural conformation which are both required for efficient processing and are involved in specific recognition of the precursors (4). The data obtained in these yeast studies are being applied to parallel experiments in Xenopus, which to date is the only existing model system for examining rRNA processing in vertebrates. A second focus is a continuation of our characterization of trans-acting factors essential for pre-rRNA processing in vertebrates. I previously demonstrated that U8 snoRNP is essential for pre-rRNA processing in Xenopus oocytes. In the absence of U8 RNA, pre-rRNA processing is inhibited and no mature rRNA accumulates (1). Mutageneis of U8 RNA indicated that sequences at the 5 prime end of U8 RNA were necessary, but not sufficient to direct pre-rRNA processing; presumably U8 RNP proteins affected the stability of the U8 RNA and the efficiency of processing (1). To better understand how the U8 RNP functions in vivo, we have been identifying proteins which specifically bind U8 RNA in vitro. We recently reported our identification of a 29 kDa protein from Xenopus ovary extracts which specifically binds U8 RNA (5). We are continuing to characterize the X29 protein. In addition we have identified a heteroheptameric complex of proteins which bind U8 snoRNA in an evolutionarily conserved octamer sequence in U8 snoRNA (6). NMR on our purified complex of proteins has identified each member of the heteroheptamer. These proteins are evolutionarily conserved and present from Archaea to human (6). Thus, U8 snoRNA function probably requires binding of the LSm complex in all organisms. We are further characterizing this protein complex and the binding site on U8 RNA to learn why binding of this protein complex is essential for in vivo function of the U8 RNP. A third focus of the lab has been an examination of snoRNA localization in oocytes and the kinetics of snoRNA-mediated pre-rRNA processing in Xenopus oocytes (7). We previously examined the kinetics of pre-rRNA processing in oocytes treated with transcriptional inhibitors to separate transcription-dependent events from those involved in processing and the data were consistent with a scenario where these snoRNAs must be present co-transcriptionally to facilitate processing. These results supports our theory that the snoRNAs act, in part, as molecular chaperones to facilitate pre-rRNA folding and we are continuing to pursue direct evidence of our working model for U8 function in vivo (1). Characterization of U8 snoRNA genes in Xenopus identified naturally occurring U8 sequence variants that are functional in vivo (8). This natural variation allowed us to identify a conserved octamer sequence in U8 snoRNA present in all vertebrate U8 snoRNAs known to date, including Xenopus, mouse, rat and human. Characterization of the octamer and identification of the protein complex that binds this sequence (6) has begun to provide insight into conserved functional mechanisms and provide additional information about the unique role of U8 snoRNP in pre-rRNA processing. Using our two model systems and taking advantage of their differences will allow us to better understand the basic mechanisms of pre-rRNA processing. With an understanding of the normal processes and components involved in ribosome biogenesis we can begin to address what aspects are abnormal in diseases like dyskeratosis congenita, scleroderma and other cell-proliferation diseases. Identification of conserved and unique cis- and trans-acting components involved in ribosome biogenesis will provide additional components to monitor in diseases involving the complex process of ribosome biogenesis.